The present invention relates to a novel gene which encodes a product which functions as a transcriptional co-activator.
By regulating the transcriptional activity of a wide variety of transcription factors, the p300/CBP family of co-activators allow diverse signals to be integrated and co-ordinated with gene expression. Significantly, p300/CBP has been implicated as a critical regulator of distinct cellular pathways, such as those leading to differentiation, cell cycle arrest and apoptosis. The molecular mechanisms that enable these processes remain unclear, although the associated histone acetyltransferase and kinase activities are likely to play an important role.
We have used a two-hybrid assay system to identify a protein which interacts with p300/CBP. The protein, which we have called JMY, has a predicted molecular weight of 110kD and lacks significant similarity to other proteins. The identification of this protein and its gene provides, inter alia, novel nucleic acids, polypeptides and related products, and novel assay methods useful in identifying novel regulators of the cell cycle.
The present invention thus provides an isolated polypeptide which comprises residues 1 to 983 of SEQ ID NO. 2 or a polypeptide having at least 70% sequence identity to SEQ ID NO. 2. The invention further provides active portions and fragments which comprises an epitope of said polypeptide.
Unless otherwise specified below, such portions and fragments are also referred to as a polypeptide of the invention.
In another aspect, the invention provides an antibody capable of binding a polypeptide of the invention, such as a monoclonal antibody.
The invention also provides an isolated nucleic acid which encodes a polypeptide of the invention, including a nucleic acid which comprises or consists essentially of nucleotides 127 to 3075 of SEQ ID NO:1 or the complement thereof. The invention further provides a nucleic acid capable of selectively hybridizing to either strand of SEQ ID NO:1 such as a nucleic acid which has at least 70% homology to SEQ ID is NO:1. Fragments of such selectively hybridizing nucleic acids are also part of the invention. Also provided by the present invention are oligonucleotides which consist essentially of from 15 to 50 contiguous nucleotides of the nucleic acids mentioned above.
Unless specified to the contrary, all the above described nucleic acids are referred to as a xe2x80x9cnucleic acidxe2x80x9d or a xe2x80x9cpolynucleotidexe2x80x9d of the invention.
The nucleic acids may be in the form of a vector, such as an expression vector wherein said nucleic acid is operably linked to a promoter heterologous to said nucleic acid. The promoter will be compatible with a desired host cell, and such host cells form a further aspect of the invention.
Nucleic acids encoding or associated with the JMY gene may be used in methods of detecting the presence or absence of said gene in a human or non-human mammalian subject, said method comprising;
(a) bringing a sample of nucleic acid from said subject into contact, under hybridizing conditions, with a polynucleotide of the invention; and
(b) determining whether said polynucleotide has been able to hybridize to a homologous sequence in said nucleic acid.
The method may be performed using a polynucleotide primer suitable for use in a polymerase chain reaction (PCR), and the determining may be performed in conjunction with a second primer using PCR such that a portion of the JMY gene is amplified.
In some instances, it the determining step may include determining the sequence of the JMY gene, when present, in the nucleic acid sample. As one alternative, restriction length fragment polymorphisms associated with the gene may be established and the assay performed with a sample which has been digested with a restriction enzyme. Another method of determining is via PCR length polymorphisms, for example through variation in the sizes of introns. Other specific means of determining hybridization are well known and routine in the art and may also be used.
The invention further provides immunological assays which comprise:
(a) bringing a body sample from said subject into contact, under binding conditions, with an antibody of the invention; and
(b) determining whether said antibody has been able to bind to a polypeptide in said sample.
We have also found that JMY is a potent coactivator of the estrogen receptor (ER). The invention thus provides assays for modulators which target the activation of the ER by JMY, optionally in the presence of p300/CBP. In another aspect, JMY also interacts with a number of transcription factors, including E2F, and these interactions provide further targets for modulators. Such modulators may influence the progression of the cell cycle.
As used herein, xe2x80x9ccomprise(s)xe2x80x9d and xe2x80x9ccomprisingxe2x80x9d are to be interpreted as xe2x80x9cinclude(s)xe2x80x9d and xe2x80x9cincludingxe2x80x9d.
The percentage homology (also referred to as identity) of DNA and amino acid sequences can be calculated using commercially available algorithms. The following programs (provided by the National Center for Biotechnology Information) may be used to determine homologies: BLAST, gapped BLAST and PSI-BLAST, which may be used with default parameters. The algorithm GAP (Genetics Computer Group, Madison, Wis.). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, the default parameters are used, with a gap creation penalty=12 and gap extension penalty=4. Use of either of the terms xe2x80x9chomologyxe2x80x9d and xe2x80x9chomologousxe2x80x9d herein does not imply any necessary evolutionary relationship between compared sequences, in keeping for example with standard use of terms such as xe2x80x9chomologous recombinationxe2x80x9d which merely requires that two nucleotide sequences are sufficiently similar to recombine under the appropriate conditions.
Another method for determining the best overall match between a SEQ ID NO:1 or SEQ ID NO:2, or portions thereof, and a query sequence is the use of the FASTDB computer program based on the algorithm of Brutlag et al (Comp. App. Biosci., 6; 237-245 (1990)). The program provides a global sequence alignment. The result of said global sequence alignment is in percent identity. Suitable parameters used in a FASTDB search of a DNA sequence to calculate percent identity are:
Matrix=Unitary, k-tuple=4, Mismatch penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty=0.05, and Window Size=500 or query sequence length in nucleotide bases, whichever is shorter. Suitable parameters to calculate percent identity and similarity of an amino acid alignment are: Matrix=PAM 150, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty=0.05, and Window Size=500 or query sequence length in nucleotide bases, whichever is shorter.